! PROJECT SUMMARY Defects in mtDNA replication and expression result in myopathies, hearing and vision loss, and other devastating disorders. The key enzyme involved in mtDNA expression, mitochondrial RNA polymerase (mtRNAP) belongs to a family of single-subunit RNAPs that is distinct from the multi-subunit cellular RNAPs. Human mtDNA contains only two promoters that share limited sequence homology; the molecular basis for recognition of these promoters remains unclear. We will use the recently obtained structures of the initiation complex to guide biochemical experiments and probe the function of mtRNAP domains involved in promoter binding, recognition, and melting. Our studies will determine the role the initiation factors play in the mechanisms of promoter recognition. We will obtain the high-resolution structures of the pre-initiation and initially transcribing initiation complexes. In the absence of transcription initiation factors mtRNAP is capable to initiate transcription on a hairpin promoter at the origin of replication OriL and thus generates replication primers. We will elucidate how mtRNAP recognizes and binds this unusual promoter and what factors affect this process. The structure of OriL-IC will be determined. Studies of the structure and function of the mtRNAP and molecular mechanisms of transcription and replication are important for understanding the regulation of mitochondrial genome expression. This, in turn, will determine our ability to influence various mitochondrial functions and as a consequence, to treat mitochondria-associated diseases. Human cells differ in the levels of mtDNA they contain. Changes in mtDNA copy number occur during normal developmental processes but have also been reported during various pathological processes and aging. At present, the mechanisms responsible for regulation of mtDNA copy number are poorly understood, impeding our ability to use mitochondria as a therapeutic target. The replication machinery in human mitochondria includes DNA polymerase gamma, TWINKLE helicase, single-stranded DNA binding protein and mtRNAP, which generates replication primers. Our recent studies identified mitochondrial transcription elongation factor, TEFM, as a major component of a molecular switch between replication and transcription in mitochondria that would allow the corresponding machineries to avoid the detrimental consequences of head-on collisions. How this switch is regulated in cells in response to different conditions is not understood. This research with determine molecular mechanism of TEFM action by biochemical and structural approaches and how changes in TEFM expression affect mtDNA copy number and mitochondrial transcription in cells during normal and stress conditions, and during cell differentiation. To elucidate the underlying mechanisms that control the set level of mtDNA, the interplay between the TEFM-dependent switch and the switch that operates at the D-loop region of mtDNA and regulates utilization of the replication intermediate, the 7S DNA, will be investigated.